CN115876919A - HPLC analysis method suitable for snake-like poison peptide - Google Patents
HPLC analysis method suitable for snake-like poison peptide Download PDFInfo
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- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 37
- 108090000765 processed proteins & peptides Proteins 0.000 title claims description 54
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- 239000002574 poison Substances 0.000 title claims description 13
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000010828 elution Methods 0.000 claims abstract description 13
- 238000010829 isocratic elution Methods 0.000 claims abstract description 8
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 8
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- OSCJHTSDLYVCQC-UHFFFAOYSA-N 2-ethylhexyl 4-[[4-[4-(tert-butylcarbamoyl)anilino]-6-[4-(2-ethylhexoxycarbonyl)anilino]-1,3,5-triazin-2-yl]amino]benzoate Chemical compound C1=CC(C(=O)OCC(CC)CCCC)=CC=C1NC1=NC(NC=2C=CC(=CC=2)C(=O)NC(C)(C)C)=NC(NC=2C=CC(=CC=2)C(=O)OCC(CC)CCCC)=N1 OSCJHTSDLYVCQC-UHFFFAOYSA-N 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to an HPLC analysis method suitable for snake-like venom peptide, which comprises the following steps: the first step is as follows: setting liquid chromatography conditions; the second step is that: formulating a mobile phase for analysis, wherein: the mobile phase A is ultrapure water; the mobile phase B is acetonitrile; the third step: the analysis program was set to isocratic elution, elution concentration: mobile phase A: 65-75%, mobile phase B: 25-35%, flow rate: 1mL/min; the fourth step: preparing a solution of one or more snake venom peptide samples to be tested, wherein the snake venom peptide samples can be single snake venom peptides or a mixture containing the snake venom peptides; the fifth step: determining one solution of the snake venom peptide sample to be detected by adopting the analysis program, and recording a chromatogram; and the like. The HPLC analysis method for snake venom peptide can continuously analyze a plurality of snake venom peptide samples and continuously measure the concentration of a plurality of snake venom peptide samples with unknown concentration.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a High Performance Liquid Chromatography (HPLC) analysis method suitable for snake venom-like peptides.
Background
Snake venom like peptides, INCI name: dipeptide diaminobutyrylbenzylamide diacetate is a synthetic tripeptide simulating the activity of snake venom toxin Waglerin 1. The tripeptide can be antagonistically combined with acetylcholine receptors, locally block nerve transmission muscle contraction information, relax facial muscles, and achieve the purposes of removing wrinkles and wrinkles by means of stroking wrinkles. Therefore, the snake venom like peptide has wide application prospect in the beauty and skin care products for the face, the neck and the hands.
HPLC analysis methods for snake venom-like peptides have been reported in the prior art. For example, CN 113720941A discloses a method for detecting a polypeptide in a skin care product raw material or a skin care product, and a method for detecting a polypeptide by high performance liquid chromatography. However, this method adds trifluoroacetic acid to the mobile phase in order to improve the peak shape of the chromatographic peak and the separation effect. This results in the end product having acid or trifluoroacetic acid residues which make the product acidic and noticeably sour, while some peptides are actually unstable under acidic conditions, affecting its shelf life. Furthermore, trifluoroacetic acid is a strong acid, which has a high requirement on the tolerance of chromatographic columns, and a reinforced chromatographic column is required because the general chromatographic column cannot bear the strong acid. Meanwhile, the retention time of the snake-like toxic peptide reported in the technical document is 14.7min, the analyzable linear range is 0.008-0.42mg/mL, and the time for one-time analysis is 36min.
In addition, along with the improvement of the product performance and safety requirements of people, higher requirements are provided for improving the separation efficiency of the chromatographic column and the accuracy of content measurement when separating and analyzing each component. When the snake-like venom peptides are analyzed, the situation that the snake-like venom peptides cannot be completely separated easily occurs, so that the content of the snake-like venom peptides cannot be accurately calculated. Meanwhile, the analysis time is shortened as much as possible in industrial production, so that the production efficiency is improved.
The theoretical plate number is one of column efficiency parameters of the chromatography and is used for quantitatively expressing the separation efficiency of the chromatographic column. At present, when HPLC analysis is carried out on snake venom-like peptide, the low theoretical plate number is a main problem in the prior art. When the lengths of the chromatographic columns are the same, the larger the theoretical plate number is, the higher the separation efficiency of the chromatographic columns is, the better the peak shape is, and accordingly, a more accurate linear graph can be obtained. Thereby providing a more accurate standard curve for the determination of the content of the snake-like poison peptide in the unknown sample.
In addition, in the prior art, under the same HLPC analysis and determination conditions, the concentration measurement range of the snake-like venom peptide is narrow, the analysis time is long, and the chromatographic conditions such as mobile phase composition and the like need to be adjusted.
Therefore, a more general and environment-friendly high performance liquid chromatography is urgently needed to be researched and searched, the method adopts an acid-free mobile phase to perform HLPC analysis on the snake-like venom peptide, can obtain a higher theoretical plate number, improves the separation efficiency of a chromatographic column, and obtains a better peak shape, so that a more accurate standard curve is provided for measuring the content of the snake-like venom peptide in a sample with unknown concentration, the content of the snake-like venom peptide in a plurality of samples with unknown concentration can be more accurately and continuously measured, and the measurement range is wider.
Disclosure of Invention
Problems to be solved by the invention
Aiming at the problems in the prior art, one of the purposes of the invention is to solve the problems that when an HPLC method is used for analyzing and measuring a snake venom peptide sample, acids such as trifluoroacetic acid and the like need to be added into a mobile phase to enhance the peak shape, so that acid substances in the sample are remained, the storage time of the product is influenced, higher requirements are put on the tolerance of a chromatographic column, and the load of the chromatographic column is increased.
The invention further aims to solve the technical problem that when an HPLC method is used for analyzing and measuring a snake venom-like peptide sample, the HPLC measurement on the snake venom-like peptide sample with unknown concentration cannot meet the requirement of higher precision due to low theoretical plate number and low separation efficiency of a chromatographic column.
It is yet another object of the present invention to solve the technical problem of narrow concentration range of sample measurement under the same HPLC chromatographic conditions.
Another purpose of the invention is to solve the problem of low production efficiency caused by too long analysis time when HPLC is used for analyzing the snake venom-like peptide.
Means for solving the problems
The present invention relates to:
1. an HPLC analysis method suitable for snake-like poison peptide, which is characterized in that:
the first step is as follows: setting liquid phase chromatographic conditions;
the second step: preparing a mobile phase for analysis, wherein; the mobile phase A is ultrapure water; the mobile phase B is acetonitrile;
the third step: the analysis program was set to isocratic elution, elution concentration: a mobile phase A: 65-75%, mobile phase B: 25-35%, flow rate: 1mL/min;
the fourth step: preparing a solution of one or more snake venom peptide samples to be tested, wherein the snake venom peptide samples can be single snake venom peptides or a mixture containing the snake venom peptides;
the fifth step: determining the solution of one snake venom peptide sample to be detected by adopting the analysis program, and recording a chromatogram map;
and a sixth step: optionally calculating the amount of snake venom peptide from the standard curve;
the seventh step: cleaning the chromatographic column by adopting a mobile phase B for 1-5 min;
the eighth step: repeating the fifth step and the seventh step, and sequentially measuring other undetected snake venom peptide-like samples to be measured.
2. The HPLC analysis method for snake venom like peptides according to item 1, wherein: the standard curve is established by preparing a standard solution of snake venom peptides and performing the assay using the liquid chromatography conditions of the first step.
3. The HPLC analysis method for snake-like venom peptides according to item 1, wherein: the wavelength range is 200-300 nm.
4. The HPLC analysis method for snake venom like peptides according to item 1 or 2, wherein: a chromatographic column with C18 packing is adopted.
5. The HPLC analysis method for snake venom like peptides according to item 1 or 2, wherein: the temperature of the chromatographic column is room temperature, the sample injection amount is 8-15 mu L, the temperature of the sample injector is ambient temperature, and the collection time of the sample to be detected is 5-10 min.
6. The HPLC analysis method for snake venom like peptides according to item 1 or 2, wherein: the concentration of the snake-like poison peptide sample is 0.01-3 mg/ml.
ADVANTAGEOUS EFFECTS OF INVENTION
The invention discovers the HPLC analysis method suitable for the snake-like poison peptide, which can accurately analyze snake-like poison peptide samples with different concentrations and accurately quantify the samples with unknown concentrations according to a standard curve. Secondly, the HPLC analysis method suitable for the snake-like venom peptide improves the peak shape under the condition of not adding trifluoroacetic acid while realizing continuous analysis and determination of a plurality of snake-like venom peptide samples, greatly improves the number of theoretical plates when the lengths of chromatographic columns are the same, thereby obtaining more accurate linear graphs, providing more accurate standard curves for determination of the content of the snake-like venom peptide in unknown samples, and further realizing more accurate continuous calibration of a plurality of snake-like venom peptide samples with unknown concentrations.
Secondly, the HPLC analysis method suitable for the snake-like poison peptide obtains higher theoretical plate number, improves the separation efficiency of the chromatographic column, thereby providing more accurate standard curve for the content determination of the snake-like poison peptide in unknown samples, and continuously measuring the content of the snake-like poison peptide in a plurality of samples with unknown concentration with higher precision.
In addition, the HPLC analysis method applicable to the snake-like poison peptide can widen the measurement concentration range of the snake-like poison peptide-containing sample under the same HPLC analysis condition.
Thirdly, the HPLC analysis method applicable to the snake-like poison peptide can shorten the analysis time, thereby greatly improving the production efficiency.
The HPLC analysis method suitable for snake-like venom peptide has simple operation, saves a large amount of time cost and economic cost when continuously analyzing and determining a plurality of snake-like venom peptide samples, and greatly simplifies the complexity of operation.
Drawings
FIG. 1 is an HPLC chart of various concentrations of snake venom peptides of example 1.
FIG. 2 is a standard graph of the concentrations of snake venom peptides of example 1.
FIG. 3 is an HPLC chart of snake venom peptide of example 2.
FIG. 4 is an HPLC chart of the snake-like peptide of comparative example 1.
FIG. 5 is an HPLC chart of snake-like venom peptide of comparative example 2.
FIG. 6 is an HPLC chart of snake-like venom peptides of comparative example 3.
FIG. 7 is an HPLC chart of snake-like venom peptides of comparative example 4.
FIG. 8 is an HPLC chart of snake-like venom peptide of comparative example 5.
FIG. 9 is an HPLC chart of snake-like venom peptide of comparative example 6.
Detailed Description
Due to the structural particularity of the snake venom like peptide, the general peptide analysis method is not suitable for the snake venom like peptide. However, when analyzing the purity, a suitable analysis method is necessary, otherwise the purity cannot be calculated according to the peak area. In order to obtain an accurate peak area, the chromatographic peak shape needs to be standard, namely, a target peak is completely separated from other peaks, the half-peak width is narrow, the peak shape is sharp, the base line is stable, and the theoretical plate number is high. Through intensive research, the invention discovers an HPLC analysis method suitable for snake venom-like peptides, which can accurately detect the content of the snake venom-like peptides and comprises the following steps:
the HPLC analysis method suitable for the snake-like venom peptide comprises the following steps:
the first step is as follows: setting liquid chromatography conditions;
the second step: preparing a mobile phase for analysis, wherein; the mobile phase A is ultrapure water; the mobile phase B is acetonitrile;
the third step: the analytical procedure was set to isocratic elution, elution concentration: mobile phase A: 65-75%, mobile phase B: 25-35%, flow rate: 1mL/min;
the fourth step: preparing a solution of one or more snake venom peptide samples to be tested, wherein the snake venom peptide samples can be single snake venom peptides or a mixture containing the snake venom peptides;
the fifth step: determining the solution of one snake venom peptide sample to be detected by adopting the analysis program, and recording a chromatogram map;
and a sixth step: optionally calculating the amount of snake venom peptide from the standard curve;
the seventh step: cleaning the chromatographic column by adopting a mobile phase B for 1-5 min;
eighth step: repeating the fifth step and the seventh step, and sequentially measuring other undetected snake venom peptide-like samples to be measured.
The standard curve was established by preparing standard solutions of various concentrations of snake venom peptides and performing the assay using the liquid chromatography conditions described above in the first step.
The mobile phase A is ultrapure water; mobile phase B was acetonitrile. The analytical procedure was isocratic elution, elution concentration: mobile phase A: 65-75%, mobile phase B:25 to 35%, preferably mobile phase a:70%, mobile phase B:30 percent. The range is selected to obtain the best analysis results. The flow rate of the mobile phase is 0.8 to 1.2mL/min, preferably 1mL/min. The flow rate is selected to be very critical, and it can shorten analysis cycle to improve the flow rate, saves time, however if the flow rate is selected inappropriately, the flow rate is too fast, then may cause two peaks to separate very closely or even overlap, can't carry out the integration, and the flow rate is too slow, may lead to few composition to be detained and lost, influences the analysis precision.
The concentration of the snake-like venom peptide sample to be detected is 0.01-3 mg/ml. Compared with the prior art, the concentration range of continuous measurement is greatly improved, in the measurement range, in order to enable the peak of the target peptide to be completely displayed, the peptide concentration is not suitable to be too high, otherwise the target peak is capped, and the judgment cannot be carried out even if a mixed peak appears at the peak top; but the concentration cannot be too low, otherwise even if there is a stray peak, the peak height is too low and the baseline is misjudged to be uneven. Therefore, the concentration of the snake-like venom peptide sample to be tested is 0.01-3 mg/ml, and more preferably 0.25-2 mg/ml.
The detection wavelength is determined by the nature of the peptide to be detected and is not influenced by the analysis conditions. However, the wavelength range is preferably 200 to 300nm, more preferably 210 to 230nm, still more preferably 260 to 280nm, and particularly preferably 215nm. The reason is that: a full wavelength scan of the snake venom peptide solution showed the strongest light absorption at 215nm, and 215nm is most preferred as the detection wavelength for HPLC analysis.
The column is a conventionally used column, preferably a column packed with C18, such as an elet ODS column.
The particle size of the packing material of the chromatography column is not particularly limited and those skilled in the art can select it according to the purpose of separation by referring to selected chromatographic conditions such as length of the chromatography column, inner diameter, selectivity for two different solutes, back pressure of the chromatography column, and the like.
The column temperature of the chromatographic column is 25 ℃, and the sample amount is 10-15 mu L, preferably 10 mu L.
The temperature of the sample injector is the ambient temperature, and the collection time of the sample to be detected is 5-10 min, preferably 5min.
The technical solution of the present invention is further described below by means of specific examples.
The invention is further illustrated by the following examples, but is not to be construed as being limited thereto, in conjunction with the accompanying drawings. It should be understood, however, that these examples are illustrative only and are not intended to limit the present invention. The raw materials used in the examples of the present invention are all those commonly used in the art, and the methods used in the examples are all those conventional in the art, unless otherwise specified.
Examples
Example 1
Instruments and conditions:
an Agilent1260InfinityII LC high performance liquid chromatograph and an OpenLabCDS2 software system are adopted; taking an Elite ODS column as a separation column, wherein the temperature of the column is 25 ℃; the ultraviolet detection wavelength is 215nm.
The experimental steps are as follows:
five groups of samples are weighed according to the amount of 0.0025g, 0.005g, 0.01g, 0.015g and 0.02g of snake venom like peptide. Dissolving the weighed snake venom peptide samples in ultrapure water to 10mL respectively to obtain snake venom peptide solutions with the concentrations of 0.25g/L, 0.5g/L, 1g/L, 1.5g/L and 2g/L respectively. Mobile phase: a: ultrapure water; b: and (3) acetonitrile.
Elution concentration: a mobile phase A:70%, mobile phase B:30 percent.
Flow rate: 1.0mL/min.
The temperature was 25 ℃.
Sample introduction amount: 10uL.
And (3) analysis program: isocratic elution; elution concentration: mobile phase A:70%, mobile phase B:30 percent.
And continuously carrying out high performance liquid chromatography analysis on the snake venom peptide-like solution with each concentration according to the chromatographic conditions, recording chromatograms, and when a test sample is replaced for testing, cleaning the chromatographic column by using acetonitrile, and carrying out operations such as chromatographic column replacement, detection wavelength change, chromatographic column temperature change and the like in the middle, and further carrying out operations such as mobile phase reconstitution, degassing and the like.
HPLC analysis of snake venom peptide solutions at concentrations of 0.25g/L, 0.5g/L, 1g/L, 1.5g/L and 2g/L, respectively, is shown in FIG. 1, and its linear standard curve is shown in FIG. 2. The retention time and the theoretical plate number of the snake-like peptide at each concentration are shown in the following table, and the relative standard deviation (% RSD) is 0.010.
The concentration-peak area linear regression equation for the snake venom peptides is shown below:
y＝ax+b
wherein: y: peak area
x. concentration of Snake venom like peptide
a:9780.81728
b:108.56559
Example 2
HPLC analysis of the snake venom peptide-like solution was carried out in the same manner as in example 1 except that the concentration of the snake venom peptide-like aqueous solution was changed to 1.25g/L, and the results are shown in FIG. 3.
Comparative example 1
Except that the mobile phase a:70%, mobile phase B:30% adjusted to mobile phase a:90%, mobile phase B: HPLC analysis of a 1.0g/L peptide solution of a snake venom like peptide sample was performed in the same manner as in example 1 except that 10%, the results are shown in FIG. 4 。
Comparative example 2
Except that the mobile phase a:70%, mobile phase B:30% adjustment to mobile phase a:80%, mobile phase B: HPLC analysis of a 1.0g/L peptide solution of a snake venom like peptide sample was performed in the same manner as in example 1 except that 20%, and the results are shown in FIG. 5 。
Comparative example 3
Except that the mobile phase a:70%, mobile phase B:30% adjustment to mobile phase a:50%, mobile phase B: HPLC analysis of a 1.0g/L peptide solution of a snake venom-like peptide sample was performed in the same manner as in example 1 except for 50%, and the results are shown in FIG. 6 。
Comparative example 4
Except that the mobile phase a: ultrapure water, 70%, mobile phase B: acetonitrile, 30% change to mobile phase a:20mM ammonium acetate aqueous solution, 90%, mobile phase B: HPLC analysis of a 1.0g/L peptide solution of a snake venom like peptide sample was performed in the same manner as in example 1 except for acetonitrile, 10%, and the results are shown in FIG. 7.
Comparative example 5
HPLC analysis was performed on a 1.0g/L peptide solution of a snake venom peptide-like sample in the same manner as in example 1, except that the analytical procedure was changed from isocratic elution to the following analytical procedure, and the results are shown in FIG. 8:
the analytical procedure was as follows:
| time min | Percentage of phase B |
| 0.1 | 5 |
| 2.5 | 5 |
| 10 | 60 |
| 12.5 | 60 |
| 12.6 | 5 |
| 15 | 5 |
Comparative example 6
HPLC analysis of a 1.0g/L peptide solution of a snake venom peptide-like sample was performed in the same manner as in example 1, except that mobile phase A was changed to methanol, mobile phase B was changed to 0.1% TFA, and the analytical procedure was changed from isocratic elution to the following analytical procedure, the results of which are shown in FIG. 9:
the analytical procedure is as follows:
| time min | Percentage of |
| 0 | 85 |
| 15 | 60 |
| 25 | 60 |
| 30 | 85 |
| 36 | 85 |
As can be seen from FIGS. 1 and 3, the peak shapes of examples 1 and 2 satisfy the definition of the typical peak shape of "selection of HPLC method in chemical analysis", the theoretical plate number is within the prescribed general range of 4000 to 40000, and the retention time is within the prescribed general range of 1 to 30 min.
Referring to FIGS. 1, 4-6, it can be seen from the results of comparing example 1 with comparative example 1, comparative example 2 and comparative example 3 that the flow matching greatly affects the analysis of the snake venom peptides, and that neither the high proportion of aqueous phase nor the low proportion of aqueous phase can achieve a good elution effect of snake venom peptides, so that a reduction of the aqueous phase to within the range of 65-75%, particularly 70%, can achieve a good elution effect of snake venom peptides, as shown in example 1.
Referring to FIGS. 1 and 7, it can be seen from the results of comparing example 1 with comparative example 4 that the addition of salt to the mobile phase also did not provide a good result in the analysis of snake-like venom peptides.
Referring to FIGS. 1 and 8, it can be seen from the results of comparing example 1 with comparative example 5 that the analysis procedure also had a large effect on the peak shape, and that the analysis procedure using gradient elution showed two absorption peaks, which adversely affected the accuracy of the concentration calculation of the snake venom-like peptide. This is probably because the snake venom like peptide was eluted when the content of phase B reached about 30% during the gradient elution, and therefore, two peaks appeared, however, as a result, the system program was erroneously judged to be impure, which affected the accuracy of the analysis, and sufficient elution of the snake venom like peptide could not be achieved.
In comparative example 6, the analytical procedure disclosed in the patent CN202111101766 was used, and as can be seen from fig. 9, in comparative example 6, although TFA was added to the mobile phase to improve the peak shape and gradient elution was used, the tailing factor was too large and the baseline was not flat although the theoretical plate number was considerable, so that the peak shape was not as excellent as the peak shape obtained in example 1.
To validate the HPLC assay method of the present invention for snake venom peptides, example 2 used a laboratory prepared snake venom peptide sample at a concentration of 1.25g/L. HPLC analytical determination was carried out by the HPLC analytical method of the present invention, and the calculated concentration of snake-like venom peptide was 1.187g/L, error: 5.04%, and the analysis precision is greatly improved.
In conclusion, the HPLC analysis method suitable for the snake venom peptide is simple to operate, and when a plurality of snake venom peptide samples are continuously analyzed and determined, a large amount of time cost and economic cost are saved, the complexity of operation is greatly simplified, the analysis precision of the snake venom peptide is improved, and the concentration range of analysis is widened.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (6)
1. An HPLC analysis method suitable for snake-like venom peptide, which is characterized in that:
the first step is as follows: setting liquid chromatography conditions;
the second step is that: preparing a mobile phase for analysis, wherein; the mobile phase A is ultrapure water; the mobile phase B is acetonitrile;
the third step: the analytical procedure was set to isocratic elution, elution concentration: a mobile phase A: 65-75%, mobile phase B: 25-35%, flow rate: 1mL/min;
the fourth step: preparing a solution of one or more snake venom peptide samples to be tested, wherein the snake venom peptide samples can be single snake venom peptides or a mixture containing the snake venom peptides;
the fifth step: determining one solution of the snake venom peptide sample to be detected by adopting the analysis program, and recording a chromatogram;
and a sixth step: optionally calculating the amount of snake venom peptide from the standard curve;
the seventh step: cleaning the chromatographic column by adopting a mobile phase B for 1-5 min;
eighth step: repeating the fifth step and the seventh step, and sequentially measuring other undetected snake venom peptide samples to be measured.
2. An HPLC analysis method suitable for snake venom peptides according to claim 1, wherein: the standard curve is established by preparing a standard solution of snake venom peptides and performing the assay using the liquid chromatography conditions of the first step.
3. The HPLC analysis method for snake venom peptides according to claim 1, wherein: the wavelength range is 200-300 nm.
4. The HPLC analysis method for snake-like venom peptides according to claim 1 or 2, wherein: a chromatographic column with C18 packing is adopted.
5. The HPLC analysis method for snake-like venom peptides according to claim 1 or 2, wherein: the temperature of the chromatographic column is room temperature, the sample injection amount is 8-15 mu L, the temperature of the sample injector is ambient temperature, and the collection time of the sample to be detected is 5-10 min.
6. The HPLC analysis method for snake-like venom peptides according to claim 1 or 2, wherein: the concentration of the snake-like poison peptide sample is 0.01-3 mg/ml.
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| CN113720941A (en) * | 2021-09-18 | 2021-11-30 | 广东丸美生物技术股份有限公司 | Detection method of polypeptide in skin care product raw material or skin care product and high performance liquid chromatography detection method of polypeptide |
| US20220034853A1 (en) * | 2020-07-30 | 2022-02-03 | Shandong Institute For Food And Drug Control | Snake venom thrombin-like enzyme marker peptide of agkistrodon halys pallas and its application in the species identification of hemocoagulase for injection |
| CN115144508A (en) * | 2022-09-02 | 2022-10-04 | 广州市乾相生物科技有限公司 | HPLC separation method suitable for multiple water-soluble peptides |
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| US20220034853A1 (en) * | 2020-07-30 | 2022-02-03 | Shandong Institute For Food And Drug Control | Snake venom thrombin-like enzyme marker peptide of agkistrodon halys pallas and its application in the species identification of hemocoagulase for injection |
| CN113720941A (en) * | 2021-09-18 | 2021-11-30 | 广东丸美生物技术股份有限公司 | Detection method of polypeptide in skin care product raw material or skin care product and high performance liquid chromatography detection method of polypeptide |
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